Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin’s one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

Line 387: SPR -spell in first on its first appearance.
Line 471-472： the templates of the two related proteins used in this homology-based approachwere they specific to any species?
Reviewer #3 (Remarks to the Author): In this study, Shenkarev and colleagues studied a class of snake three-finger toxins with nicotinic acetylcholine receptors. They focus their attention to a weak non-conventional neurotoxin WTX from Naja kaouthia. This type of neurotoxin binds with weak affinity to mammalian nAChRs (tens μM), including the alpha7 nAChR. To validate their approach, the authors first determined the cryo-EM structure of the alpha7-AChBP with alpha-BgTx and compared it to the known X-ray crystal structure of this complex. In this context it should be noted that the authors use the term "alpha7-ECD" whereas the protein really is the alpha7-AChBP. This set aside, the authors went on to determine the cryo-EM structure of the alpha7-AChBP/WTX complex. Detailed molecular insight into the toxinreceptor interaction is precluded by the low resolution of the data (5.61 Angstrom). This resolution is insufficient to reveal secondary structures and this a major weakness in the study. The authors proceeded with a combination of complementary techniques to further explore the toxin-receptor interaction. These techniques include NMR spectroscopy of the free WTX molecule, combined with previous mutagenesis data and in silico docking. Despite the low resolution of the data, the authors suggest a binding mode in which the WTX toxin interacts at a site that also includes the membrane. Finally, the authors employ molecular dynamics simulations and mutagenesis data to further explore this interaction mode.
In my opinion, the low resolution of the cryo-EM for the WTX-AChBP complex precludes the detailed interpretation of the toxin binding mode. The authors engage in using a multi-method approach to reveal the correct binding mode, including mutagenesis, docking and MD simulations. However, my feeling is that the quality of the data just do not justify the conclusions made.
Reviewer #4 (Remarks to the Author): This article implements multiple biophysical methods to elucidate the binding mechanism of the noconventional toxin WXT to the α7-nAChR receptor. The authors utilized a soluble extra cellular domain of α7-nAChR for structural studies by cryoEM, NMR interaction studies of WXT with model membranes and combined these data in-silico studies to derive a α7-nAChR/WTX structure consistent with the experimental data. Although the cryoEM data are low resolution the resultant models are consistent. Overall, the studies are rigorous and provide insight into the binding of non-conventional toxins to α7-nAChR and merit publication after minor revisions and points to address below.
1. The authors used the muscle type structure to build the full length α7-nAChR model. While a highresolution structure of α7-nAChR is now available. This is only pointed out in the methods but should be more clearly stated in the main txt so the reader is aware. A related point is the statement that the RMSD values of "3.0 Å for the whole structure, 2.7 Å for the ECD, 2.4 Å for the TM domain, and 2.5 Å for the ligand-binding site" between the homology model and the actual recent structure "indicate that our model is precise enough and is a reliable starting point for MD calculations". This reviewer is unaware of any objective RMSD criteria that indicates a model is precise enough for starting MD calculations. If there is one, then the authors should reference accordingly.
2. The lipid binding studies of the WTX mutants are limited and somewhat surprising. None of the positive charged mutations alter affinity, however the negative charge mutation combined with a single positive mutation abolishes binding. It would be most informative to include the single E21A mutant in these studies to validate this result.
3. The study would benefit from performing the in-silico approach with the α-Bgtx complex as this structure is known. This would provide further confidence in the results from the α7-nAChR/WTX complex.
4. The supplement would benefit from including a cryoEM data table. By convention defocus values are positive numbers and not negative as stated in the text.
5. There are several minor typographical errors that could be corrected.
Reviewer #5 (Remarks to the Author): The manuscript from Shenkarev and coworkers shows the interaction of a weak three-finger toxin with α7-ECD by cryomicroscopy. They show that the local interaction differs from that of α-bungarotoxin, a toxin with a high affinity to this receptor. They were able to complement this information using some mutants, RMN studies, molecular modeling, and some previously published data. The article presents some relevant new information on the binding mechanism of the WTX molecule to the α7-nAChR especially related to the relevance of the toxin binding to the membrane. Although I am not an expert in cryomicroscopy, I was able to apprehend the applied methodology and the conclusions reached by the authors. Their initial motivation, as described in the introduction section, was to use the WTX as a tool to understand the interaction of molecules such as that from the Ly6/uPAR family, which can bind nAChR with low affinity. Although this is an interesting idea, one question arose: why they did not use the human three-fingers molecules themselves? I think that it would be interesting to clarify which are the common structural features, especially that related to the binding site and to the membrane-binding characteristics.
The authors stated that line 359 "Altogether, this allows to consider WTX as the prototypical neuromodulator from the toxin kingdom" It would be of interest to verify the sequence similarities of the endogenous three fingers proteins from snakes in order to consider this a plausible hypothesis. Minor comments: The abstract does not have any consideration on human three-fingers proteins. The introduction section should consider only the novelty of revealing other mechanisms of interaction between two correlated toxins... The discussion of human three-finger could be present only in the discussion section. Otherwise, the authors should explain better why they are not using human molecules for this study. Results are well described, and figures are well picked and of good quality. Methods are well described and I believe that is reproducible. The manuscript is of interest to Toxinology and Neuroscience.

Reviewer #1 (Remarks to the Author):
The manuscript describes the interaction of beta-bungarotoxin and WTX with α7-ECD. The authors have carefully designed the experiments and collected the data presented in the manuscript. They have used Cryo-EM data combined with NMR data, the define the WTX-α7-ECD structure. This structure differs from canonical interaction of beta-bungarotoxin. The authors propose distinct 'membrane catalysis' mechanism for non-conventional neurotoxins.
Although most parts of the manuscript are well written, some parts require a thorough rewriting. The unusual use of words makes it difficult to follow the logic at times.

Answer:
We tried to do our best to improve the English language of our manuscript and rewrote some sections.

Reviewer #1
Although it is clear that WTX binds to phospholipids, it is important to show experimentally the interaction of the phospholipids enhances the interaction with α7-ECD.

Answer:
Thank you for this valuable remark. The experimental confirmation of this idea is not easy. At present we even could not envisage the method how to do that. Especially if we have soluble variant of α7-ECD without membrane interacting part. The way how to conduct such experiments on the full-length membrane-embedded receptor is also non-evident. Therefore, we used computer simulations (deltaG calculations with umbrella sampling) to show this. We calculated the energy of interaction of WTX with the full-length alpa7 receptor embedded into the membrane, with the extracellular domain of the receptor, and with the lipid bilayer in the absence of the receptor. Data obtained confirmed the significant contribution of the membrane (up to 40%) into the free energy of WTX binding to the receptor. These data were included in the revised manuscript (Figure 7).

Reviewer #2 (Remarks to the Author):
Authors reported an interesting work where cryo-EM was used to study the interactions of α7-nAChR extracellular ligand-binding domain (α7-ECD) with its ligands, applying α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR) and WTX, a non-conventional three-finger toxin which has not been widely studied. The methodology is sound and robust, and the findings are new with potential to benefit future studies involving α7-nAChR as this is present in many biological systems and is a promising therapeutic target for cognitive disfunctions and cancer therapy. The manuscript is well written and can be considered for publication following minor revision. Suggestions for improvement are given below.